Our overall research program addresses the need for much more efficient methods of synthesizing and manufacturing peptide- and protein-based drugs. The objective of this application is the development, using novel peptide coupling/ligation chemistries, of two new syntheses of enfuvirtide (below) that are far more sustainable than the extant enfuvirtide synthesis. The aims for this project are: 1. Modify the existing fragment condensation synthesis of enfuvirtide to generate the N-terminal (1- 16) fragment and the C-terminal (27-36) fragment via solution-phase synthesis using recently demonstrated N-carboxyanhydride (NCA) couplings (enhanced by solvophobic techniques) and serine ligation methods. 2. Develop a de novo synthesis of enfuvirtide using sub-segments from Aim 1 assembled by serine ligations to form the serine-rich N-terminal (1-11) and C-terminal (30-36) fragments, followed by serine ligation of these fragments to a central (12-29) fragment. The expected outcomes of this work include: procedures that enable preparative production of the enfuvirtide drug substance in fewer operations with reduced use of reagents and solvents and the generation of significantly less chemical waste; demonstration of novel NCA couplings and serine ligations in the context of a challenging molecular target that will endorse the strategy for application to other peptide therapeutics. Synthesis of enfuvirtide is relevant to human health because it is a valuable and unique antiretroviral drug for the treatment of HIV-1 infection. However, the cost of enfuvirtide therapy (related to its high manufacturing costs) is prohibitive, so it is typically held in reserve for salvage therapy in patients with multi-drug resistant HIV. The impact of the work will be based on both the potential to drive down the cost of a valuable drug therapy as well as the provision of guidance to others aiming to develop new methods for peptide drug preparation. This project will utilize the following methods: organic synthesis, peptide synthesis, and solid-phase synthesis.